The present invention relates to a method of introducing at least one single-chain insulin analog protein or a gene encoding a single-chain insulin analog (SIA) into at least one mammalian tissue for use in treating diabetes in the mammalian host. The present invention also relates to the single-chain insulin analog and a recombinant vector construct comprising the gene encoding SIA.
The cure of diabetes has long been sought using several different approaches, including islet transplantation, regeneration of xcex2 cells and insulin gene therapy (Levine, F. and Leibowitz, G. Towards gene therapy of diabetes mellitus. Mol. Med. Today 5, 165-171 (1999)). However, the permanent remission of type 1 diabetes has not yet been satisfactorily achieved.
WO96/34882 discusses making single chain insulin with high bioactivity, but does not disclose the single chain insulin analog of the invention.
There remains a very real and substantial need for a method of introducing at least one gene encoding a single-chain insulin analog to at least one cell of a mammalian host in vitro or in vivo, for use in treating the mammalian host suffering from diabetes.
The present invention has met the hereinbefore described need.
A method of introducing at least one gene encoding a product into at least one cell of a mammalian tissue for use in treating a mammalian host is provided in the present invention. This method includes employing recombinant techniques to produce a DNA vector molecule containing the gene coding for the product and introducing the DNA vector molecule containing the gene coding for the product into the tissue cell. The DNA vector molecule can be any DNA molecule capable of being delivered and maintained within the target cell or tissue such that the gene encoding the product of interest can be stably expressed. The DNA vector molecule preferably utilized in the present invention is either a viral or plasmid DNA vector molecule. This method preferably includes introducing the gene encoding the product into the cell of the mammalian tissue for a therapeutic use.
An object of the invention is to provide a single-chain insulin analog compound of formula (I) having the properties of greater insulin receptor binding activity than proinsulin and less insulin receptor binding activity than insulin:
B chain-X-A chainxe2x80x83xe2x80x83(I)
wherein:
B and A chains are the human insulin chains, respectively, or functional analogs thereof; and
X is a joining peptide of from 5 to 18 amino acids.
In the above compound, preferably, X may be from 6 to 9 amino acids.
Also, in the above compound, when X has the formula Ulxe2x80x94Znxe2x80x94Ymxe2x80x94Zlxe2x80x94Un, the following limitations may be placed:
U is an arginine or lysine residue;
Z is an amino acid residue;
Y is a peptide;
l is an integer of 2-n;
n is an integer of 0, 1 or 2; and
m is an integer of 2 to 5
In this compound, Z may be glycine; and Y may be glycine-proline-glycine. Furthermore, Z may be glycine; and Y may be alanine-proline-glycine-aspartic acid-valine. Alternatively, Z may be glycine; and Y may be tyrosine-proline-glycine-aspartic acid-valine. Further, Z may be glycine; and Y may be histidine-proline-glycine-aspartic acid-valine.
Another object of the invention is to provide a polynucleotide encoding the single-chain insulin analog described above. Another embodiment of the invention includes a recombinant vector comprising the polynucleotide that encodes the single chain insulin analog described above. The vector may be a plasmid or a virus. If a virus, preferably, it is adeno-associated virus. Moreover, it is preferred that the promoter be inducible. More preferably, the promoter may be regulated by glucose. Even more preferably, the promoter is a pyruvate kinase gene promoter. Most preferably, the promoter is the hepatocyte-specific L-type pyruvate kinase gene promoter.
The invention is also directed to a cell line transformed with the above-described vector.
Another embodiment of the invention is directed to a method for treating a patient suffering from diabetes comprising:
a) generating a recombinant viral or plasmid vector comprising a polynucleotide encoding a single-chain insulin analog operatively linked to a promoter; and
b) introducing said recombinant viral or plasmid vector to said patient, such that expression of said polynucleotide within said patient results in remission of diabetes.
Preferably, the viral vector is adeno-associated virus, and the promoter is an inducible promoter. Preferably, the promoter is regulated by glucose. In the method described above, preferably the dosage of said viral vector is at least about 1011 viral particles. Preferably, the treatment method is accomplished by using a vector that is introduced to the patient through the cell line comprising the single chain insulin analog described above.
The present invention is also directed to a method for treating a patient suffering from diabetes comprising administering the single chain insulin analog compound described above to a patient in need thereof. Preferably, the diabetes is type I diabetes.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.